A new oxygen modification cyclooctaoxygen binds to nucleic acids as sodium crown complex.

BACKGROUND: Oxygen exists in two gaseous and six solid allotropic modifications. An additional allotropic modification of oxygen, the cyclooctaoxygen, was predicted to exist in 1990. METHODS: Cyclooctaoxygen sodium was synthesized in vitro from atmospheric oxygen, or catalase effect-generated oxygen, under catalysis of cytosine nucleosides and either ninhydrin or eukaryotic low-molecular weight RNA. Thin-layer chromatographic mobility shift assays were applied on specific nucleic acids and the cyclooctaoxygen sodium complex. RESULTS: We report the first synthesis and characterization of cyclooctaoxygen as its sodium crown complex, isolated in the form of three cytosine nucleoside hydrochloride complexes. The cationic cyclooctaoxygen sodium complex is shown to bind to nucleic acids (RNA and DNA), to associate with single-stranded DNA and spermine phosphate, and to be essentially non-toxic to cultured mammalian cells at 0.1-1.0mM concentration. CONCLUSIONS: We postulate that cyclooctaoxygen is formed in most eukaryotic cells in vivo from dihydrogen peroxide in a catalase reaction catalyzed by cytidine and RNA. A molecular biological model is deduced for a first epigenetic shell of eukaryotic in vivo DNA. This model incorporates an epigenetic explanation for the interactions of the essential micronutrient selenium (as selenite) with eukaryotic in vivo DNA. GENERAL SIGNIFICANCE: Since the sperminium phosphate/cyclooctaoxygen sodium complex is calculated to cover the active regions (2.6%) of bovine lymphocyte interphase genome, and 12.4% of murine enterocyte mitotic chromatin, we propose that the sperminium phosphate/cyclooctaoxygen sodium complex coverage of nucleic acids is essential to eukaryotic gene regulation and promoted proto-eukaryotic evolution.

Isolation and identification of compounds from Kalanchoe pinnata having human alphaherpesvirus and vaccinia virus antiviral activity.

CONTEXT: Kalanchoe pinnata (Lam.) Pers. (Crassulaceae) is a succulent plant that is known for its traditional antivirus and antibacterial usage. OBJECTIVE: This work examines two compounds identified from the K. pinnata plant for their antivirus activity against human alphaherpesvirus (HHV) 1 and 2 and vaccinia virus (VACV). MATERIALS AND METHODS: Compounds KPB-100 and KPB-200 were isolated using HPLC and were identified using NMR and MS. Both compounds were tested in plaque reduction assay of HHV-2 wild type (WT) and VACV. Both compounds were then tested in virus spread inhibition and virus yield reduction (VYR) assays of VACV. KPB-100 was further tested in viral cytopathic effect (CPE) inhibition assay of HHV-2 TK-mutant and VYR assay of HHV-1 WT. RESULTS: KPB-100 and KPB-200 inhibited HHV-2 at IC50 values of 2.5 and 2.9 µg/mL, respectively, and VACV at IC50 values of 3.1 and 7.4 µg/mL, respectively, in plaque reduction assays. In virus spread inhibition assay of VACV KPB-100 and KPB-200 yielded IC50 values of 1.63 and 13.2 µg/mL, respectively, and KPB-100 showed a nearly 2-log reduction in virus in VYR assay of VACV at 20 µg/mL. Finally, KPB-100 inhibited HHV-2 TK- at an IC50 value of 4.5 µg/mL in CPE inhibition assay and HHV-1 at an IC90 of 3.0 µg/mL in VYR assay. DISCUSSION AND CONCLUSION: Both compounds are promising targets for synthetic optimization and in vivo study. KPB-100 in particular showed strong inhibition of all viruses tested.

Synthesis and biological evaluation of epidithio-, epitetrathio-, and bis-(methylthio)diketopiperazines: synthetic methodology, enantioselective total synthesis of epicoccin G, 8,8'-epi-ent-rostratin B, gliotoxin, gliotoxin G, emethallicin E, and haematocin and discovery of new antiviral and antimalarial agents.

An improved sulfenylation method for the preparation of epidithio-, epitetrathio-, and bis-(methylthio)diketopiperazines from diketopiperazines has been developed. Employing NaHMDS and related bases and elemental sulfur or bis[bis(trimethylsilyl)amino]trisulfide (23) in THF, the developed method was applied to the synthesis of a series of natural and designed molecules, including epicoccin G (1), 8,8'-epi-ent-rostratin B (2), gliotoxin (3), gliotoxin G (4), emethallicin E (5), and haematocin (6). Biological screening of selected synthesized compounds led to the discovery of a number of nanomolar antipoliovirus agents (i.e., 46, 2,2'-epi-46, and 61) and several low-micromolar anti- Plasmodium falciparum lead compounds (i.e., 46, 2,2'-epi-46, 58, 61, and 1).

NITD-688, a pan-serotype inhibitor of the dengue virus NS4B protein, shows favorable pharmacokinetics and efficacy in preclinical animal models.

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility. The lead compound, NITD-688, showed strong potency against all four serotypes of DENV and demonstrated excellent oral efficacy in infected AG129 mice. There was a 1.44-log reduction in viremia when mice were treated orally at 30 milligrams per kilogram twice daily for 3 days starting at the time of infection. NITD-688 treatment also resulted in a 1.16-log reduction in viremia when mice were treated 48 hours after infection. Selection of resistance mutations and binding studies with recombinant proteins indicated that the nonstructural protein 4B is the target of NITD-688. Pharmacokinetic studies in rats and dogs showed a long elimination half-life and good oral bioavailability. Extensive in vitro safety profiling along with exploratory rat and dog toxicology studies showed that NITD-688 was well tolerated after 7-day repeat dosing, demonstrating that NITD-688 may be a promising preclinical candidate for the treatment of dengue.

(-)-Carbodine: enantiomeric synthesis and in vitro antiviral activity against various strains of influenza virus including H5N1 (avian influenza) and novel 2009 H1N1 (swine flu).

Enantiomerically pure cyclopentyl cytosine [(-)-carbodine 1] was synthesized from d-ribose and evaluated for its anti-influenza activity in vitro in comparison to the (+)-carbodine, (+/-)-carbodine and ribavirin. (-)-Carbodine 1 exhibited potent antiviral activity against various strains of influenza A and B viruses.

Development of a respiratory disease model for enterovirus D68 in 4-week-old mice for evaluation of antiviral therapies.

Enterovirus D68 (EV-D68) is a non-polio enterovirus that affects the respiratory system and can cause serious complications, especially in children and older people with weakened immune systems. As an emerging virus, there are no current antiviral therapies or vaccines available. Our goal was to develop a mouse model of human EV-D68 infection that mimicked the disease observed in humans and could be used for evaluation of experimental therapeutics. This is the first report of a respiratory disease model for EV-D68 infection in mice. We adapted the virus by 30 serial passages in AG129 mice, which are deficient in IFN- α/ß and -γ receptors. Despite a lack of weight loss or mortality in mice, lung function measured by plethysmography, showed an increase in enhanced pause (Penh) on days 6 and 7 post-infection. In addition, as virus adapted to mice, virus titer in the lungs increased 50-fold, and the pro-inflammatory cytokines MCP-1 and RANTES increased 15-fold and 2-fold in the lung, respectively. In addition, a time course of mouse-adapted EV-D68 infection was determined in lung, blood, liver, kidney, spleen, leg muscle, spinal cord and brain. Virus in the lung replicated rapidly after intranasal inoculation of adapted virus, 106 CCID50/mL by 4 h and 108.3 CCID50/mL by 24 h. Virus then spread to the blood and other tissues, including spinal cord and brain. This mouse model for EV-D68 infection includes enhanced pause (Penh) as an indicator of morbidity, and viremia, virus titers and proinflammatory cytokines in the lung, and lung histopathology as indicators of disease. Our mouse-adapted virus has a similar antiviral profile to the original isolate as well as another respiratory picornavirus, rhinovirus-14. This model will be valuable in evaluating experimental therapies in the future.

Interferon alfacon 1 inhibits SARS-CoV infection in human bronchial epithelial Calu-3 cells.

The primary targets for SARS-CoV infection are the epithelial cells in the respiratory and intestinal tract. The angiotensin-converting enzyme 2 (ACE-2) has been identified as a functional receptor for SARS-CoV. ACE-2 has been shown to be expressed at the apical domain of polarized Calu-3 cells. In this report, interferon alfacon 1 was examined for inhibitory activities against SARS-CoV on human lung carcinoma epithelial Calu-3 cell line and the other three African green monkey kidney epithelial cell lines. Interferon alfacon 1 demonstrated significant antiviral activity in neutral red uptake assay and virus yield reduction assay. The data might provide an important insight into the mechanism of pathogenesis of SARS-CoV allowing further development of antiviral therapies for treating SARS infections.

Is the anti-psychotic, 10-(3-(dimethylamino)propyl)phenothiazine (promazine), a potential drug with which to treat SARS infections? Lack of efficacy of promazine on SARS-CoV replication in a mouse model.

Phenothiazine and derivatives were tested for inhibition of SARS-CoV replication. Phenothiazine slightly inhibited SARS-CoV replication in a neutral red (NR) uptake assay. Adding a propylamino group to give promazine reduced virus yields (VYR assay) with an EC(90)=8.3+/-2.8 microM, but without selectivity. Various substitutions in the basic phenothiazine structure did not promote efficacy. Phenazine ethosulfate was the most potent compound by VYR assay (EC(90)=6.1+/-4.3 microM). All compounds were toxic (IC(50)=6.6-74.5 microM) except for phenoxathiin (IC(50)=858+/-208 microM) and 10-(alpha-diethylamino-propionyl) phenothiazine.HCl (IC(50)=195+/-71.2 microM). Consequently, none were selective inhibitors of SARS-CoV replication (SI values <1-3.3 microM). These data portended the poor efficacy of promazine in a SARS-CoV mouse lung replication model. Intraperitoneal treatment with promazine using a prophylactic (-4h)/therapeutic regimen of 1, 10, or 50mg/(kg day) did not reduce virus lung titers at day 3, yet prolonged virus replication to 14 days. Similar therapeutic promazine doses were not efficacious. Thus, promazine did not affect SARS-CoV replication in vitro or in vivo, nor were any other phenothiazines efficacious in reducing virus replication. Therefore, treating SARS infections with compounds like promazine is not warranted.

Novel 3-sulphonamido-quinazolin-4(3H)-one derivatives: microwave-assisted synthesis and evaluation of antiviral activities against respiratory and biodefense viruses.

We designed and synthesized novel 2,3-disubstituted quinazolin-4(3H)-ones by microwave technique and characterized them by spectral analysis. Synthesized compounds were screened for cytotoxicity and for antiviral activity against influenza A (H1N1, H3N2 and H5N1), severe acute respiratory syndrome corona, dengue, yellow fever, Venezuelan equine encephalitis (VEE), Rift Valley fever, and Tacaribe viruses in cell culture. A neutral red uptake assay was used to determine 50% virus-inhibitory concentrations (EC50) of test compounds and their 50% cytotoxicity concentration (CC50) in uninfected Madin-Darby canine kidney, Vero, and Vero 76 cells; selectivity indices (ratio of CC50 to EC50) were derived from the data. The compound 4-(6,8-dibromo-4-oxo-2-phenyl quinazolin-3(4H)-yl)-N-(4,5-dimethyloxazol-2yl) benzenesulphonamide 15 inhibited the replication of avian influenza (H5N1) virus (EC50 = 8.4 microg/ml, CC50 > 100 microg/ml, SI > 11.9) as did 4-(6-bromo-4oxo-2phenylquinazolin-3(4H)-yl) benzene]sulphonamide 5 (EC50 = 3 microg/ml, CC50 = 32 microg/ml, SI = 11). Compound 5 was also moderately active against VEE and Tacaribe viruses. The methodology described in this report is applicable for rapid synthesis of many compounds with potential antiviral properties.

Effect of oral gavage treatment with ZnAL42 and other metallo-ion formulations on influenza A H5N1 and H1N1 virus infections in mice.

Avian influenza H5N1 infections can cause severe, lethal human infections. Whether influenza A virus treatments effectively ameliorate avian influenza H5N1 human infections is uncertain. The research objective was to evaluate the efficacy of novel zinc and other metallo-ion formulations in two influenza A mouse models. Mice infected with influenza A/Duck/MN/1525/81 (H5N1) virus were treated orally 48 h before virus exposure and then twice daily for 13 days with ZnAL42. The optimal dosing regimen for ZnAL42 was achieved at 17.28 mg/kg 48 h prior to virus exposure, twice daily for 7 days. The survival rate was 80% compared with 10% in the untreated control group and a 100% survival rate with ribavirin (75 mg/kg/day, twice a day for 5 days, beginning 4 h before virus exposure). ZnAL42 treatment significantly lessened the decline in arterial oxygen saturation (SaO2; P < 0.001). This regimen was also well tolerated by the mice. Manganese and selenium formulations were not inhibitory to virus replication when given therapeutically. Mice were also infected with influenza A/NWS/33 (H1N1) virus and were treated 48 h before virus exposure with three dosages of ZnAL42 (8.64, 1.46 or 0.24 mg/kg/day). Treatment was by oral gavage twice daily for 13 days. The highest dose of ZnAL42 was significantly inhibitory to the virus infection as seen by prevention of deaths and lessening of decline in SaO2. The data suggest that the prophylactic use of ZnAL42 is effective against avian influenza H5N1 or H1N1 virus infection in mice and should be further explored as an option for treating human influenza virus infections.

Evaluation of cell viability dyes in antiviral assays with RNA viruses that exhibit different cytopathogenic properties.

Studies were conducted to determine the performance of four dyes in assessing antiviral activities of compounds against three RNA viruses with differing cytopathogenic properties. Dyes included alamarBlue® measured by absorbance (ALB-A) and fluorescence (ALB-F), neutral red (NR), Viral ToxGlo™ (VTG), and WST-1. Viruses were chikungunya, dengue type 2, and Junin, which generally cause 100, 80-90, and 50% maximal cytopathic effect (CPE), respectively, in Vero or Vero 76 cells Compounds evaluated were 6-azauridine, BCX-4430, 3-deazaguanine, EICAR, favipiravir, infergen, mycophenolic acid (MPA), ribavirin, and tiazofurin. The 50% virus-inhibitory (EC50) values for each inhibitor and virus combination did not vary significantly based on the dye used. However, dyes varied in distinguishing the vitality of virus-infected cultures when not all cells were killed by virus infection. For example, VTG uptake into dengue-infected cells was nearly 50% when visual examination showed only 10-20% cell survival. ALB-A measured infected cell viability differently than ALB-F as follows: 16% versus 32% (dengue-infected), respectively, and 51% versus 72% (Junin-infected), respectively. Cytotoxicity (CC50) assays with dyes in uninfected proliferating cells produced similar CC50 values for EICAR (1.5-8.9µM) and MPA (0.8-2.5µM). 6-Azauridine toxicity was 6.1-17.5µM with NR, VTG, and WST-1, compared to 48-92µM with ALB-A and ALB-F (P<0.001). Curiously, the CC50 values for 3-deazaguanine were 83-93µM with ALB-F versus 2.4-7.0µM with all other dyes including ALB-A (P<0.001). Overall, ALB minimized the toxicities detected with these two inhibitors. Because the choice of dyes affected CC50 values, this impacted on the resulting in vitro selectivity indexes (calculated as CC50/EC50 ratio).

Enhancement of the infectivity of SARS-CoV in BALB/c mice by IMP dehydrogenase inhibitors, including ribavirin.

Because of the conflicting data concerning the SARS-CoV inhibitory efficacy of ribavirin, an inosine monophosphate (IMP) dehydrogenase inhibitor, studies were done to evaluate the efficacy of ribavirin and other IMP dehydrogenase inhibitors (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), mizoribine, and mycophenolic acid) in preventing viral replication in the lungs of BALB/c mice, a replication model for severe acute respiratory syndrome (SARS) infections (Subbarao, K., McAuliffe, J., Vogel, L., Fahle, G., Fischer, S., Tatti, K., Packard, M., Shieh, W.J., Zaki, S., Murphy, B., 2004. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in the respiratory tract of mice. J. Virol. 78, 3572-3577). Ribavirin given at 75 mg/kg 4 h prior to virus exposure and then given twice daily for 3 days beginning at day 0 was found to increase virus lung titers and extend the length of time that virus could be detected in the lungs of mice. Other IMP dehydrogenase inhibitors administered near maximum tolerated doses using the same dosing regimen as for ribavirin were found to slightly enhance virus replication in the lungs. In addition, ribavirin treatment seemed also to promote the production of pro-inflammatory cytokines 4 days after cessation of treatment, although after 3 days of treatment ribavirin inhibited pro-inflammatory cytokine production in infected mice, significantly reducing the levels of the cytokines IL-1alpha, interleukin-5 (IL-5), monocyte chemotactic protein-1 (MCP-1), and granulocyte-macrophage colony stimulating factor (GM-CSF). These findings suggest that ribavirin may actually contribute to the pathogenesis of SARS-CoV by prolonging and/or enhancing viral replication in the lungs. By not inhibiting viral replication in the lungs of infected mice, ribavirin treatment may have provided a continual source of stimulation for the inflammatory response thought to contribute to the pathogenesis of the infection. Our data do not support the use of ribavirin or other IMP dehydrogenase inhibitors for treating SARS infections in humans.

Evaluation of immunomodulators, interferons and known in vitro SARS-coV inhibitors for inhibition of SARS-coV replication in BALB/c mice.

Compounds approved for therapeutic use and in vitro inhibitors of severe acute respiratory syndrome coronavirus (SARS-CoV) were evaluated for inhibition in the mouse SARS-CoV replication model. A hybrid interferon, interferon alpha (IFN-alpha) B/D, and a mismatched double-stranded (ds) RNA interferon (IFN) inducer, Ampligen (poly I:poly C124), were the only compounds that potently inhibited virus titres in the lungs of infected mice as assessed by CPE titration assays. When mice were dosed intraperitoneally (i.p.) with IFN-alpha B/D once daily for 3 days beginning 4 h after virus exposure, SARS-CoV replication in the lungs of infected mice was reduced by 1 log10 at 10,000 and 32,000 IU; at the highest dose of 100,000 IU, virus lung titres were below detectable limits. Ampligen used i.p. at 10 mg/kg 4 h prior to virus exposure also reduced virus lung titres to below detectable limits. Nelfinavir, beta-D-N4-hydroxycytidine, calpain inhibitor VI, 3-deazaneplanocin A and Alferon (human leukocyte IFN-alpha-n3) did not significantly reduce lung virus titres in mice. Anti-inflammatory agents, chloroquine, amodiaquin and pentoxifylline, were also inactive in vivo, suggesting that although they may be useful in ameliorating the hyperinflammatory response induced by the virus infection, they will not significantly reduce the replication of the virus, the inducer of inflammatory response. Thus, anti-inflammatory agents may only be useful in treating virus lung infections if used in combination with agents that inhibit virus replication. In summary, the data suggest that induction of IFN by mismatched dsRNA or actual treatment with exogenous IFN-alpha can inhibit SARS-CoV replication in the lungs of mice.

Susceptibilities of enterovirus D68, enterovirus 71, and rhinovirus 87 strains to various antiviral compounds.

Compounds were evaluated for antiviral activity in rhabdomyosarcoma (RD) cells against a recent 2014 clinical isolate of enterovirus D68 (EV-D68), a 1962 strain of EV-68D, rhinovirus 87 (RV-87, serologically the same as EV-D68), and enterovirus 71 (EV-71). Test substances included known-active antipicornavirus agents (enviroxime, guanidine HCl, pirodavir, pleconaril, and rupintrivir), nucleobase/nucleoside analogs (3-deazaguanine and ribavirin), and three novel epidithiodiketopiperazines (KCN-2,2'-epi-19, KCN-19, and KCN-21). Of these, rupintrivir was the most potent, with 50% inhibition of viral cytopathic effect (EC50) and 90% inhibition (EC90) of virus yield at 0.0022-0.0053 µM against EV-D68. Enviroxime, pleconaril and the KCN compounds showed efficacy at 0.01-0.3 µM; 3-deazaguanine and pirodavir inhibited EV-D68 at 7-13 µM, and guanidine HCl and ribavirin were inhibitory at 80-135 µM. Pirodavir was active against EV-71 (EC50 of 0.78 µM) but not against RV-87 or EV-D68, and all other compounds were less effective against EV-71 than against RV-87 and EV-D68. The most promising compound inhibiting both virus infections at low concentrations was rupintrivir. Antiviral activity was confirmed for the ten compounds in virus yield reduction (VYR) assays in RD cells, and for enviroxime, guanidine HCl, and pirodavir by cytopathic effect (CPE) assays in A549, HeLa-Ohio-1, and RD cells. These studies may serve as a basis for further pre-clinical discovery of anti-enterovirus inhibitors. Furthermore, the antiviral profiles and growth characteristics observed herein support the assertion that EV-D68 should be classified together with RV-87.

The Iminosugar UV-4 is a Broad Inhibitor of Influenza A and B Viruses ex Vivo and in Mice.

Iminosugars that are competitive inhibitors of endoplasmic reticulum (ER) α-glucosidases have been demonstrated to have antiviral activity against a diverse set of viruses. A novel iminosugar, UV-4B, has recently been shown to provide protection against lethal infections with dengue and influenza A (H1N1) viruses in mice. In the current study, the breadth of activity of UV-4B against influenza was examined ex vivo and in vivo. Efficacy of UV-4B against influenza A and B viruses was shown in primary human bronchial epithelial cells, a principal target tissue for influenza. Efficacy of UV-4B against influenza A (H1N1 and H3N2 subtypes) and influenza B was demonstrated using multiple lethal mouse models with readouts including mortality and weight loss. Clinical trials are ongoing to demonstrate safety of UV-4B and future studies to evaluate antiviral activity against influenza in humans are planned.

Error-prone replication of West Nile virus caused by ribavirin.

Ribavirin has been reported to cause error-prone replication and viral extinction in RNA viruses. The antiviral activity of ribavirin against West Nile virus (WNV) was evaluated in various cell lines to select a model in which mutagenic effects could be studied. The antiviral activity was greatest in HeLa cells as compared to CV-1, L929, Vero, or MA-104 cells. WNV was also passaged sequentially in cell monolayers treated with ribavirin to determine whether cumulative mutations could lead to viral extinction in these cell lines. The virus was abrogated in HeLa cells after 4 passages, while high viral titers persisted after many passages in other cells. A molecular clone of WNV was propagated in HeLa cells treated with 15 microg/mL ribavirin, and sequencing of viral genome segments revealed significant increases in transition mutations, demonstrating that ribavirin induced error-prone replication. The relative infectivity of viral RNA synthesized in the presence of ribavirin was shown to be reduced compared with untreated controls. These data support the hypothesis that error catastrophe is one of the modes of action for ribavirin against WNV.

Treatment of West Nile virus-infected mice with reactive immunoglobulin reduces fetal titers and increases dam survival.

The objectives of this study were to determine if injection of West Nile virus (WNV) into timed-pregnant mice would result in fetal infection and if administration of WNV-reactive immunoglobulin would increase dam survival and reduce fetal viral titers. Dams injected on 7.5 days post-coitus (dpc) had detectable viral titers in the placenta 10.5dpc with a mean titer of 10(4.9) 50% cell-culture infectious doses per gram of tissue (CCID(50)/g tissue). The mean placental titer increased to 10(8.6)CCID(50)/g tissue at 12.5dpc. Infectious virus was detectable 12.5dpc in 10 of 10 fetuses with a mean titer of 10(7.5)CCID(50)/g tissue. Treatment of dams (challenged with WNV on 7.5dpc) with WNV-reactive human immunoglobulin (Ig) on 8.5 and 9.5dpc resulted in a significant reduction of virus in fetuses as compared with non-reactive human Ig-treated females on 12.5dpc (P< or =0.001). Treatment also resulted in survival of dams to term. Treatment of dams with WNV-reactive human Ig on 12.5 and 13.5dpc also resulted in reduction of viral titer on 14.5dpc, indicating that later treatment may also be efficacious. This suggests that Ig treatment may be useful in treating fetal WNV infection in women.

Modeling hamsters for evaluating West Nile virus therapies.

A hamster model infected with a New York crow brain isolate of West Nile virus (WNV) was characterized for evaluating potential antiviral therapies. Older hamsters (7-11 weeks old) had a lower mortality of approximately 50% and more apparent disease signs as compared to >90% mortality in younger hamsters and mice. Disease signs such as limb strength, lacrimation, front limb tremors, somnolence, and deficiencies in neurological responses were noted at different times after viral injection. Weight loss was a marker for WNV disease signs, whereas, the ability to climb up an inclined ramp was associated with whether the animals survived the disease or died. Infectious WNV assays performed on tissues from hamsters during development of the infection indicated that viral titers peaked first in plasma, but that titers were eventually highest in kidney tissue. Viral titers achieved maximal levels in brain tissue on 6 dpi, which was 1-2 days before strong neurological signs and death started to occur. Maximal spleen and plasma titers were achieved sooner in young hamsters as compared with older hamsters, which correlated with increased susceptibility. To test the hypothesis that older hamsters would be more sensitive for identifying antiviral effects, Infergen, a consensus human interferon-alpha highly active against WNV in cell culture, was administered subcutaneously to older and younger hamsters beginning on 2 dpi. The effects of Infergen on weight change, survival, and climbing ability of infected animals were more apparent in older hamsters than in younger hamsters. The use of older hamsters is another WNV-infectious model, in addition to mice, for evaluating potential antiviral therapies.

Effect of interferon-alpha and interferon-inducers on West Nile virus in mouse and hamster animal models.

The recent West Nile virus (WNV) outbreak in the United States has increased the need to identify effective therapies. Studies were conducted in cell culture and in rodent animal models to determine the efficacy of interferon-alpha (IFN-alpha), interferon (IFN) inducers and ribavirin, alone or in combination with IFN, in treating WNV. Intraperitoneal injection of IFN-alpha B/D (qd for 7 days), polyI-polyC(12)U [Ampligen (every other day for 7 days)] and topically applied imiquimod (qd for 7 days), administered from 1 day before viral challenge, were effective in protecting, respectively, 100%, 100% and 70% of BALB/c mice from mortality induced by subcutaneous injection of WNV. When IFN-alpha B/D or Ampligen treatments were delayed to 4-6 h before viral challenge in mice, efficacy was greatly diminished. Infected Syrian golden hamsters treated with interferon alphacon-1 (Infergen) and Ampligen 4-6 h before viral challenge gained more weight and had a greater survival than saline-treated animals. A combination study of subcutaneously administered Infergen (5 to 0.05 microg/kg/day) and ribavirin (75 to 7.5 mg/kg/day) in >7 week old hamsters demonstrated that Infergen was slightly efficacious in reducing mortality and disease signs; however, it was not synergistic in its antiviral effects when combined with ribavirin. Ribavirin treatment alone increased mortality of infected hamsters. The reduced mortality correlated with reduced plasma viraemia. Since WNV-infected patients have already been treated with IFN and ribavirin and future clinical trials have been suggested, this first report of IFN alone or in combination with ribavirin in WNV-infected animal models might provide useful information for subsequent treatment of patients.

Influenza Virus H1N1 inhibition by serine protease inhibitor (serpin) antithrombin III.

Endogenous serine protease inhibitors (serpins) are anti-inflammatory mediators with multiple biologic functions. Serpins are also part of the early innate immune response to viral infection that includes mannose binding lectins, soluble CD14, defensins and antimicrobial peptides. Recently, serpin antithrombin III (ATIII) was shown to have broad-spectrum antiviral activity against HIV, HSV and HCV. We tested ATIII's antiviral activity against a variety of influenza virus strains. In our studies we found strong in vitro inhibition of influenza virus A H1N1 isolates. Our data also demonstrate that ATIII potency was more than 100-fold that of ribavirin. We also found that inhibition was dependent on viral hemagglutinin with decreasing efficacy in the order of H1N1 > H3N2 > H5N1 >> Flu B. In vivo efficacy is currently still lacking demonstrating need for more advanced delivery methods for this biomolecule. Understanding how ATIII regulates influenza virus inhibition may reveal new avenues for therapeutic interventions.